Label-Free Identification of Spore-Forming Bacteria Using Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy.

Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm-1. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.

Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

Visualization of intracellular lipid metabolism in brown adipocytes by time-lapse ultra-multiplex CARS microspectroscopy with an onstage incubator.

We visualized a dynamic process of fatty acid uptake of brown adipocytes using a time-lapse ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging system with an onstage incubator. Combined with the deuterium labeling technique, the intracellular uptake of saturated fatty acids was traced up to 9 h, a substantial advance over the initial multiplex CARS system, with an analysis time of 80 min. Characteristic metabolic activities of brown adipocytes, such as resistance to lipid saturation, were elucidated, supporting the utility of the newly developed system.

Visualizing intra-medulla lipids in human hair using ultra-multiplex CARS, SHG, and THG microscopy.

We performed label-free imaging of human-hair medulla using multi-modal nonlinear optical microscopy. Intra-medulla lipids (IMLs) were clearly visualized by ultra-multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging. Two groups of IMLs were found: second harmonic generation (SHG) active and inactive. By combining SHG analysis with CARS, the two groups were identified as free fatty acids and wax esters, respectively.

Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB.

For many years, scientists have been looking for specific biomarkers associated with cancer cells for diagnosis purposes. These biomarkers mainly consist of proteins located at the cell surface (e.g. the TrkB receptor) whose activation is associated with specific metabolic modifications. Identification of these metabolic changes usually requires cell fixation and specific dye staining. MCARS microspectroscopy is a label-free, non-toxic, and minimally invasive method allowing to perform analyses of live cells and tissues. We used this method to follow the formation of lipid droplets in three colorectal cancer cell lines expressing TrkB. MCARS images of cells generated from signal integration of CH2 stretching modes allow to discriminate between lipid accumulation in the endoplasmic reticulum and the formation of cytoplasmic lipid droplets. We found that the number of the latter was related to the TrkB expression level. This result was confirmed thanks to the creation of a HEK cell line which over-expresses TrkB. We demonstrated that BDNF-induced TrkB activation leads to the formation of cytoplasmic lipid droplets, which can be abolished by K252a, an inhibitor of TrkB. So, MCARS microspectroscopy proved useful in characterizing cancer cells displaying an aberrant lipid metabolism.

Multiplex coherent anti-Stokes Raman scattering highlights state of chromatin condensation in CH region.

Coherent Raman microscopy has become a powerful tool in label-free, non-destructive and fast cell imaging. Here we apply high spectral resolution multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy in the high wavenumber region to the study of the cell cycle. We show that heterochromatin - the condensed state of chromatin - can be visualised by means of the vibrational signature of proteins taking part in its condensation. Thus, we are able to identify chromosomes and their movement during mitosis, as well as structures like nucleoli and nuclear border in interphase. Furthermore, the specific organization of the endoplasmic reticulum during mitosis is highlighted. Finally, we stress that MCARS can reveal the biochemical impact of the fixative method at the cellular level. Beyond the study of the cell cycle, this work introduces a label-free imaging approach that enables the visualization of cellular processes where chromatin undergoes rearrangements.

Characterization of Intra/Extracellular Water States Probed by Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopic Imaging.

Detailed knowledge of the water status in living organisms is crucial for understanding their physiology and pathophysiology. Here, we developed a technique to spectroscopically image water at high resolution using ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy equipped with a supercontinuum light source. This system allows for the visualization of a wide spectrum of CARS signals from the fingerprint to the end of O-H stretching at a spectral resolution of ∼10 cm-1. Application of the system to living mammalian cells revealed a spectral red shift of the O-H stretching vibrational band inside compared to outside the cells, suggesting the existence of stronger hydrogen bonds inside the cells. Furthermore, potential changes in spectra were examined by adding mannitol to the extracellular solution, which increases the osmolality outside the cells and thereby induces dehydration of the cells. Under this treatment, the red shift of the O-H stretching band was further enhanced, revealing the effects of mannitol on water states inside the cells. The methodology developed here should serve as a powerful tool for the chemical imaging of water in living cells in various biological and medical contexts.

Fast epi-detected broadband multiplex CARS and SHG imaging of mouse skull cells.

We present a bimodal imaging system able to obtain epi-detected mutiplex coherent anti-Stokes Raman scattering (M-CARS) and second harmonic generation (SHG) signals coming from biological samples. We studied a fragment of mouse parietal bone and could detect broadband anti-Stokes and SHG responses originating from bone cells and collagen respectively. In addition we compared two post-processing methods to retrieve the imaginary part of the third-order nonlinear susceptibility related to the spontaneous Raman scattering.

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation.

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.

Spectro-temporal shaping of supercontinuum for subnanosecond time-coded M-CARS spectroscopy.

A supercontinuum laser source was designed for multiplex-coherent anti-Stokes Raman scattering spectroscopy. This source was based on the use of a germanium-doped standard optical fiber with a zero dispersion wavelength at 1600 nm and pumped at 1064 nm. We analyzed the nonlinear spectro-temporal interrelations of a subnanosecond pulse propagating in a normal dispersion regime in the presence of a multiple Raman cascading process and strong conversion. The multiple Raman orders permitted the generation of a high-power flat spectrum with a specific nonlinear dynamics that can open the way to subnanosecond time-coded multiplex CARS systems.

Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source.

Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red(®) are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three-photon ones.

Raman optical activity spectroscopy by visible-excited coherent anti-Stokes Raman scattering.

We developed a Raman optical activity (ROA) spectroscopic system with visible-excited coherent anti-Stokes Raman scattering (CARS). A supercontinuum within the visible region was generated with a photonic crystal fiber pumped with both 532 and 1064 nm excitation, generating a multiplexed CARS-ROA spectrum covering the whole fingerprint region. In visible excitation, the CARS-ROA spectrum of (-)-ß-pinene shows a higher contrast ratio of the chirality-induced signal to the achiral background than that of the previously reported near-infrared CARS-ROA spectrum.

Multimodal imaging of living cells with multiplex coherent anti-stokes raman scattering (CARS), third-order sum frequency generation (TSFG) and two-photon excitation fluorescence (TPEF) using a nanosecond white-light laser source.

The subnanosecond "white-light laser" source has been applied to multimodal, multiphoton, and multiplex spectroscopic imaging (M(3) spectroscopic imaging) with coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and two-photon excitation fluorescence (TPEF). As the proof-of-principle experiment, we performed simultaneous imaging of polystyrene beads with TSFG and TPEF. This technique is then applied to live cell imaging. Mouse L929 fibroblastic cells are clearly visualized by CARS, TSFG, and TPEF processes. M(3) spectroscopic imaging provides various and unique cellular information with different image contrast based on each multiphoton process.

Near-infrared supercontinuum laser beam source in the second and third near-infrared optical windows used to image more deeply through thick tissue as compared with images from a lamp source.

With the use of longer near-infrared (NIR) wavelengths, image quality can be increased due to less scattering (described by the inverse wavelength power dependence 1/λ(n) where n ≥ 1 ) and minimal absorption from water molecules. Longer NIR windows, known as the second (1100 nm to 1350 nm) and third (1600 to 1870 nm) NIR windows are utilized to penetrate more deeply into tissue media and produce high-quality images. An NIR supercontinuum (SC) laser light source, with wavelengths in the second and third NIR optical windows to image tissue provides ballistic imaging of tissue. The SC ballistic beam can penetrate depths of up to 10 mm through tissue.

Multimodal and multiplex spectral imaging of rat cornea ex vivo using a white-light laser source.

We applied our multimodal nonlinear spectral imaging microscope to the measurement of rat cornea. We successfully obtained multiple nonlinear signals of coherent anti-Stokes Raman scattering (CARS), third-order sum frequency generation (TSFG), and second harmonic generation (SHG). Depending on the nonlinear optical processes, the cornea tissue was visualized with different image contrast mechanism simultaneously. Due to white-light laser excitation, multiplex CARS and TSFG spectra were obtained. Combined multimodal and spectral analysis clearly elucidated the layered structure of rat cornea with molecular structural information. This study indicates that our multimodal nonlinear spectral microscope is a promising bioimaging method for tissue study. Multimodal nonlinear spectral images of rat cornea at corneal epithelium and corneal stroma in the in-plane (XY) direction. With use of the combinational analysis of different nonlinear optical processes, detailed molecular structural information is available without staining or labelling.

Electronically resonant third-order sum frequency generation spectroscopy using a nanosecond white-light supercontinuum.

Third-order sum frequency generation (TSFG) is one of the third-order nonlinear optical processes, and has the generation mechanism analogous to third harmonic generation (THG). By using a white-light supercontinuum, we can obtain broadband multiplex TSFG spectra. In the present study, we developed an electronically resonant TSFG spectrometer, and applied it to obtain TSFG spectra of hemoproteins. Analyzed TSFG ratio spectra clearly showed the resonant enhancement attributable to the electronic state of hemoproteins. This is a promising method for the imaging of electronic states of molecules inside living cells or tissues.

Surfactant uptake dynamics in mammalian cells elucidated with quantitative coherent anti-stokes Raman scattering microspectroscopy.

The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%). By using an isotope labeled surfactant having CD bonds, surfactant uptake dynamics in living cells has been traced in detail. The simultaneous CARS imaging of the cell itself and the internalized surfactant has shown that the surfactant molecules is first accumulated inside a CHL cell followed by a sudden leak of cytosolic components such as proteins to the outside of the cell. This finding indicates that surfactant uptake occurs prior to the cell lysis, contrary to what has been believed: surface adsorption of surfactant molecules has been thought to occur first with subsequent disruption of cell membranes. Quantitative CARS microspectroscopy enables us to determine the molecular concentration of the surfactant molecules accumulated in a cell. We have also investigated the effect of a drug, nocodazole, on the surfactant uptake dynamics. As a result of the inhibition of tubulin polymerization by nocodazole, the surfactant uptake rate is significantly lowered. This fact suggests that intracellular membrane trafficking contributes to the surfactant uptake mechanism.

Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy.

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

Observation of Raman optical activity by heterodyne-detected polarization-resolved coherent anti-Stokes Raman scattering.

We report the first observation of Raman optical activity (ROA) by coherent anti-Stokes Raman scattering. Thanks to the more freedom of polarization configurations in coherent anti-Stokes Raman scattering than in spontaneous Raman spectroscopy, the contrast ratio of the chiral signal to the achiral background has been improved markedly. For (-)-ß-pinene, it is 2 orders of magnitude better than that in the reported spontaneous ROA measurement. This is also the first measurement of ROA signal using a pulsed laser source.

Flow cytometer based on triggered supercontinuum laser illumination.

Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this multiparametric technique is currently used to differentiate blood cells subpopulations. The choice of excitation wavelengths matching fluorochrome spectra (it is currently the opposite) and the use of a broader range of fluorochromes can be made by taking advantage of a filtered supercontinuum white light source. In this study, we first wished to validate the use of a specific triggered supercontinuum laser in a flow cytometer based on white light scattering and electric sizing on human blood cells. Subsequently, to show the various advantages of this attractive system, using scattering effect, electrical detections, and fluorescence analysis, we realized cells sorting based on DNA/RNA stained by thiazole orange. Discrimination of white blood cells is efficiently demonstrated by using a triggered supercontinuum-based flow cytometer operating in a "one cell-one shot" configuration. The discriminated leukocyte populations are monocytes, lymphocytes, granulocytes, immature granulocytes, and cells having a high RNA content (monoblasts, lymphoblasts, and plasma cells). To the best of our knowledge, these results constitute the first practical demonstration of flow cytometry based on triggered supercontinuum illumination. This study is the starting point of a series of new experiments fully exploiting the spectral features of such a laser source. For example, the large flexibility in the choice of the excitation wavelength allows to use a larger number of fluorochromes and to excite them more efficiently. Moreover, this work opens up new research directions in the biophotonics field, such as the combination of coherent Raman spectroscopy and flow cytometry techniques.